Three-dimensional (3D) random access laser scanning technologies have great importance in performing measurements on biological specimens (including scanning, imaging, detection, excitation, etc.) e.g. imaging biological structures or mapping fluorescent markers of cell surface receptors or performing measurements such as uncaging/photosimulation, FRET (Fluorescence resonance energy transfer), FLIM (Fluorescence lifetime imaging), etc.
Commonly used 3D laser scanning microscopes are either confocal microscopes or multi-photon (two-photon) microscopes. In the confocal microscope technology a pinhole is arranged before the detector to filter out light reflected from any other plane than the focus plane of the microscope objective. Thereby it is possible to image planes lying in different depths within a sample (e.g. a biological specimen).
Two-photon laser scanning microscopes use a laser light of lower energy of which two photons are needed to excite a flourophore in a quantum event, resulting in the emission of a fluorescence photon, which is then detected by a detector. The probability of a near simultaneous absorption of two photons is extremely low requiring a high flux of excitation photons, thus two-photon excitation practically only occurs in the focal spot of the laser beam, i.e. a small ellipsoidal volume having typically a size of approximately 300 nm×300 nm×1000 nm. Generally a femtosecond pulsed laser is used to provide the required photon flux for the two-photon excitation, while keeping the average laser beam intensity sufficiently low.
When applying either of the above-mentioned technologies the conventional way to perform the scanning in 2D or 3D is to move the sample stage e.g. via stepping motors; however this is complicated to implement when using submersed specimen chambers or when electrical recording is performed on the biological specimen with microelectrodes. Accordingly, in the case of analysing biological specimens it is often preferred to move the focus spot of the laser beam instead of moving the specimen. In confocal and multi-photon microscopes 2D scanning of the specimen can be achieved by deflecting the laser beam to scan different points of a focal plane (XY plane). This scanning is conventionally achieved by deflecting the laser beam within a given focal plane (XY plane) via mechano-optical deflecting means such as deflecting mirrors mounted on galvanometric scanners. Changing the depth of focus during measurement (or scanning along the z axis) can be achieved for example by displacing the objective along its optical axis (Z axis) e.g. using a piezo-positioner.
The inertia of the mechanical scanning components used in the conventional setups (i.e. the scanning mirrors and the microscope objective) presents certain limitations with regard to the achievable scanning speed, since the scanning components need to be physically moved in order to perform 3D scanning.
Rapid acousto-optic deflectors (AOD) have been proposed as an alternative to the conventional mechanic solutions.
Kaplan et al. (“Acousto-optic lens with very fast focus scanning”, OPTICS LETTERS/Vol. 26, No. 14/Jul. 15, 2001)) proposed an acousto-optic lens made up of two AODs with counter propagating acoustic waves locked in phase, to achieve purely focus shift without lateral moving of the beam. Changing the focus of the acousto-optic lens was achieved by changing the sweep rate of the acoustic frequencies through the acousto-optic devices.
In U.S. Pat. No. 7,227,127 the above principle is made use of in order to provide 3D scanning. The focus of the beam can be moved in space in a diamond like structure by using four acousto-optic deflectors, two for both lateral directions (X and Y). Lateral scanning is a result of the acoustic frequency differences of the two AODs diffracting in the same lateral direction, whereas depth focusing (i.e. focus shift along the microscope optical axis named Z axis) is achieved by changing the sweep rate of the acoustic frequencies in the same AODs. Thus the focus point can be quasi independently adjusted in the X-Z and Y-Z planes, where Z is the longitudinal direction corresponding to the optical axis of the device. This also leads to strong astigmatism, when the frequency sweep rates in the X and Y deflecting units are not perfectly matched.
Furthermore, there are various problems associated with the state of the art AOD 3D scanning technology such as spatial and temporal dispersion, especially when applied in combination with multi-photon scanning technologies.
Short femtosecond pulses applied in multi-photon technologies necessarily imply larger spectral width, i.e. a larger spread of wavelengths exists in a shorter pulse leading to greater spatial dispersion (chromatic aberration). Temporal dispersion (i.e. elongation of the pulse in time) is caused by the fact that different wavelengths of light travel at different speeds through the AOD.
Prior art AOD systems eliminate spatial and temporal dispersion to a more or less extent by applying complicated dispersion compensating elements.
It has been shown in many previous papers and patents that the highest angle range and resolution can be obtained with AODs using optically anisotropic diffraction, namely a special configuration where the acousto-optic diffraction is achieved with a shear acoustic wave and the polarization of the first order diffracted beam is nearly perpendicular to that of the incident-beam. This configuration works well in a number of optically anisotropic materials with slow shear acoustic modes like PbMnO4 or TeO2. In this patent we propose a new configuration of a scanner preferably comprising TeO2 deflectors operating in anisotropic configuration to obtain the highest possible angular (optical) resolution at the output.
When an anisotropic AOD deflects an incoming laser beam, the polarisation of the first order beam is rotated by 90 degrees compared to the incoming laser beam and the zero order undeflected transmitted beam.
It is commonly accepted that the bandwidth obtainable with an anisotropic AOD is considerably higher when using extraordinary incident light instead of ordinary incident light due to the interaction geometry. In order to operate all Bragg cells with extraordinary light it is proposed in WO 2008/6032061 to provide half wave plates between the AODs of each AOD pair operating in the X-Z plane and in the Y-Z plane, respectively, for rotating the polarisation of the first order beam deflected by the first AOD of the pair, thus providing extraordinary incident light for the second AOD of the pair as well. The use of half wave plates has many drawbacks: the wave plates are angle sensitive limiting the usable divergence and propagation angle of the passing beams, moreover wave plates are wavelength sensitive, thus it may cause bandwidth reduction and material dispersion in case of the high bandwidth femtosecond pulses used in multi-photon technologies.
As an alternative, the same document proposes arranging the AODs of the two pairs alternately, i.e. the first X-Z AOD is followed by the first Y-Z AOD, followed by the second X-Z AOD, followed by the second Y-Z AOD. As it is commonly known an AOD operating in the X-Z plane and an AOD operating in the Y-Z plane require perpendicularly polarised light with respect to each other, thus the rotated polarisation of the first order deflected beam exiting the X-Z AOD is suitable for the consequent Y-Z AOD and vice versa.
However, the inventors of the present invention have found that the high bandwidth commonly associated with the use of extraordinary incident light is obtainable only in a limited incident angle range. The angle tolerance is usually three-five times less then the angle range provided by the same deflector in the deflected (scanned) beam. Therefore if two similar anisotropic deflectors are arranged consecutively so that both deflect in the same plane (e.g. X-Z plane) and the polarization is rotated between them so as both operate with extraordinary polarized incident beam, the overall deflection angle range is automatically reduced by three-five times despite of the difference in the center acoustic frequency between the cells. This reduces both the scanned X-Y range in the focal plane and the focal length variation range within the X-Z and Y-Z planes, respectively.
FIG. 1 shows the relation between the diffraction efficiency of an anisotropic AOD operated with extraordinary polarized incident beam and the deflection angle at constant acoustic power. Note that considerable diffraction efficiency can be obtained approximately in a 1.7 deg range. FIG. 2. shows a typical dependence of the diffraction efficiency of an anisotropic AOD operating with extraordinary polarized incident beam on the acoustic frequency and incidence angle. It is obvious that the incidence angle range over which considerable diffraction efficiency is achieved depends on the acoustic frequency and is about 0.5 to 1 degrees wide.
When both AODs operate with extraordinary incidence at constant electric power (a very common and simple driving scheme) and constant optical input, the dependence of the output optical intensity on the frequencies of both AOD's will be as shown in FIG. 3. The deep minimum in the band is caused by the second order diffraction characteristic to these types of deflectors. The effect of the second order diffraction appears in the case of the higher bandwidth obtainable with the extraordinary polarized optical incidence and causes a deep minimum in the overall bandwidth. This avoids the effective use of this portion of the frequency band for e.g. focusing, since the serious output optical intensity variation with frequency in this area causes distortion of the focused spot. However, this effect is reduced when the acoustic (more precisely the driving electric) power is reduced, but in this case the first order efficiency is also reduced. Compensation of this effect by driving with frequency dependent power is also limited because of this reason.
On the other hand, when the first AOD of a pair deflecting in the same transversal plane is operating with ordinary incident wave, and the second one with extraordinary incident one, the arrangement dispenses with the need for a half wave plate. The ordinary optical incidence in the first AOD provides a smaller output angle range than the extraordinary incidence, but still bigger than the input angle range accepted by the second AOD, as shown in FIG. 4. and compared with FIG. 2.
FIG. 5 shows the diffraction bandwidth in the X-Z plane with two consecutive anisotropic AODs, wherein the first AOD operates with ordinary, the second AOD operates with extraordinary polarized incident beam. When compared with FIG. 3 it is immediately evident that here the second order diffraction has a reduced effect because it is apparent only in the second AOD, where extraordinary optical incidence is applied. This means that this configuration has practically a bigger bandwidth than the first configuration where both AOD's were operated with extraordinary polarized optical inputs.
It is an object of the invention to overcome the problems associated with the prior art laser scanning microscopes.
In particular, it is an object of the invention to provide an anisotropic AOD arrangement, which eliminates the need of half wave plates, and at the same time allows for maximal bandwidth.
It is a further object of the invention to effectively combine the AODs in order to obtain 3D scanning with optimum spatial and temporal resolution, meaning optimum spatial and temporal dispersion when light with broad wavelength spectrum (e.g. very short pulse) is used together with effective compensation for the strong imaging aberrations caused by the acousto-optic devices.